Improving the thermostability and acid resistance of Rhizopus oryzae α-amylase by using multiple sequence alignment based site-directed mutagenesis.

Higher thermostability or acid resistance for fungal α-amylase will help to improve the sugar-making process and cut down the production costs. Here, the thermostability or acid resistance of Rhizopus oryzae α-amylase (ROAmy) was significantly enhanced by site-directed evolution based on multiple sequence alignment (MSA) method. For instance, compared with the wild-type ROAmy, the optimum temperature of mutants G136D and A144Y was increased from 50 to 55 °C, whereas for mutants V174R and I276P, the optimum temperature was increased from 50 to 60 °C. The optimum pH of mutants G136D and A144Y shifted from 5.5 to 5.0, whereas for mutants V174R and T253E, the optimum pH changed from 5.5 to 4.5. The results showed that mutant V174R had a 2.52-fold increase in half-life at 55 °C, a 2.55-fold increase in half-life at pH 4.5, and a 1.61-fold increase in catalytic efficiency (kcat /Km ) on soluble starch. The three-dimensional model simulation revealed that changes of hydrophilicity, hydrogen bond, salt bridge, or rigidity observed in mutants might mainly account for the improvement of thermostability and acid resistance. The mutants with improved catalytic properties attained in this work may render an accessible and operable approach for directed evolution of fungal α-amylase aimed at interesting functions.